The invention relates to cathepsin O2 proteins, nucleic acids, and antibodies.
The cathepsins belong to the papain superfamily of cysteine proteases. Cysteine or thiol proteases contain a cysteine residue, as well as a histidine and an asparagine, at the active site responsible for proteolysis. This superfamily also has a glutamine at the oxy-anion hole.
Recent work has implicated cysteine proteases in binding to DNA with putative transcription factor activity (Xu et al., J. Biol. Chem. 269(33):21177-21183 (1994)), and as a long term immunosuppressor (Hamajima et al., Parasite Immunology 16:261 (1994)).
To date, a number of cathepsins have been identified and sequenced from a number of animals. For example, cathepsin S has been cloned from rat (Petanceska et al., J. Biol. Chem. 267:26038-20643 (1992)), bovine (Wiederanders et al., FEBS Lett. 286:189-192 (1991)) and humans (Wideranders et al., J. Biol. Chem. 267:13708-13713 (1992); and Shi et al., J. Biol. Chem. 267:7258-7262 (1992)). Cathepsin L has been cloned from humans, rat, mouse and chicken (Gal et al. Biochem. J., 253:303-306 (1988); Ishidoh et al., FEBS Lett. 223:69-73 (1987); Joseph et al., J. Clin. Invest. 81:1621-1629 (1988); Ritonja et al., FEBS Lett. 283:329-331 (1991)). Cathepsin H has been cloned from human and rat (Fuchs et al., Biol. Chem. Hoppe-Seyler 369-375 (1988); Fuchs et al., Nucleic Acid Res. 17:9471 (1989); Whittier et al., Nucleic Acid Res. 15:2515-2535 (1987)). Cathepsin B has been cloned from human and mouse (Ferrara et al., FEBS Lett. 273:195-199 (1990); Chan et al., Proc. Natl. Acad. Sci. USA 83:7721-7725 (1986)).
A cysteine protease from rabbit osteoclasts was recently cloned, and is structurally related to cathepsins L and S. Tezuka et al., J. Biol. Chem. 269(2):1106 (1994).
Cathepsins are naturally found in a wide variety of tissues. For example, cathepsin L is found in tissues including heart, brain, placenta, lung, skeletal muscle, kidney, liver, testis and pancreas. Cathepsin S is found in lung, liver, spleen and skeletal muscle.
Cathepsins have been implicated in a number of disease conditions. For example, enzymes similar to cathepsins B and L are released from tumors and may be involved in tumor metastasis. Cathepsin L is present in diseased human synovial fluid and transformed tissues. Similarly, the release of cathepsin B and other lysosomal proteases from polymorphonuclear granulocytes and macrophages is observed in trauma and inflammation. Cathepsins have been implicated in arthritis. In addition, cathepsins are found in abnormally high amounts in several tumor cell lines.
Cysteine proteases have also been implicated in bone remodeling. Bone remodeling is a process coupling bone formation and bone resorption, and is part of bone growth. Bone resorption includes demineralization and degradation of extracellular matrix proteins (Delaisse et al., Biochem. J. 279:167-174 (1991)). Type I collagen constitutes ninety-five percent of the organic matrix (Krane et al., in Scientific American Medicine (Rubensttein, E., and Federman, D. D., eds) Vol. 3, 15 Rheumatism, XI Bone Formation and Resorption, pp. 1-26, Scientific American, Inc. New York. In addition to the interstitial collagenase, the lysosomal cysteine proteases cathepsins B and L are thought to be involved in osteoclastic bone resorption (Delaisse et al., 1991, supra). Both enzymes are present in the lysosomes as well as in the acidified extracellular resorption lacuna of the osteoclast (Goto et al., Histochemistry 99, 411-414(1993)) and both proteases display the in vitro ability to degrade collagen Type I at acidic pH (Maciewicz et al., Collagen Rel. Res. 7, 295-304 (1987), Delaisse et aL, (1991), supra). Cysteine protease inhibitors, such as E-64 and leupeptin, have been shown to prevent osteoclastic bone resorption (Delaisse et al., Bone 8, 305-313 (1987), Everts et al., Calcif. Tissue Int. 43, 172-178 (1988)). Cathepsin L is considered to be one of the main proteases involved in collagen degradation in bone (Maciewiecz et al., Biochem. J. 256, 433-440 (1988); Kakegawa et al., FEBS Lett. 321, 247-250 (1993)).
The solid state of bone material is due to the low solubility of hydroxyapatite and other calcium-phosphate bone salts at physiological pH, but bone may break down at acidic pH.
Osteoclasts are multinucleate cells that play key roles in bone resorption. Attached to the bone surface, osteoclasts produce an acidic microenvironment in a tightly defined junction between the specialized osteoclast border membrane and the bone matrix, thus allowing the localized solubilization of bone matrix. This in turn facilitates the protolysis of demineralized bone collagen.
It is thought that the collagenolytic action of cysteine proteases is exerted preferentially in the most acidic part of the bone resorption lacuna close to the ruffled border at a pH around 3.5 or 4.5, whereas the Zn-containing collagenases are more active in the neutral environment at the interface between the demineralized and mineralized matrix (Delaisse et al., supra, (1991)). Besides cathepsins L and B, a variety of cathepsin L- and B-like activities may participate in collagenolytic bone degradation. Page et al. Biochim. Biophys. Acta 1116, 57-66 (1992) isolated multiple forms of cathepsin B from osteoclastomas. These have an acidic pH optimum and the ability to degrade soluble and insoluble Type I collagen. Delaisse et al., 1991, supra, identified a 70 kDa thiol-dependent protease in bone tissue which is also capable of degrading Type I collagen.
Cysteine protease inhibitors have been shown to inhibit osteoclastic bone resorption by inhibiting degradation of collagen fibers. Cathepsins B, L, N and S can degrade type-I collagen at acidic pH. Three cathepsin-type proteases have been isolated from mouse calvaria; putative cathepsins B and L, and a cathepsin L-like:protease (Delaisse et al., Biochem. J. 279:167 (1991). However, it is still unclear as to what cysteine proteases are actually produced by osteoclasts. Recently, a cDNA encoding a novel human cysteine protease was cloned independently by several groups (Shi et al., FEBS Lett. 357, 129-134 (1995),
Inaoka et al., Biochem. Biophys. Res. Commun. 206, 89-96 (1995); Brxc3x6mme and Okamoto, Biol. Chem. Hoppe-Seyler 376, 379-384 (1995)) and named cathepsin O, cathepsin K, and cathepsin O2, respectively.
It is an object of the present invention to provide for a new class of recombinant cathepsins, cathepsin O2, and variants thereof, and to produce useful quantities of these cathepsin O2 proteins using recombinant DNA techniques.
It is a further object of the invention to provide recombinant nucleic acids encoding cathepsin O2 proteins, and expression vectors and host cells containing the nucleic acid encoding the cathepsin O2 protein.
An addition object of the invention is to provide poly- and monoclonal antibodies for the detection of the presence of cathepsin O2 and diagnosis of conditions associated to cathepsin O2.
A further object of the invention is to provide methods for producing the cathepsin O2 proteins.
In accordance with the foregoing objects, the present invention provides recombinant cathepsin O2 proteins, and isolated or recombinant nucleic acids which encode the cathepsin O2 proteins of the present invention. Also provided are expression vectors which comprise DNA encoding a cathepsin O2 protein operably linked to transcriptional and translational regulatory DNA, and host cells which contain the expression vectors.
Additional aspect of the present invention provides methods for producing cathepsin O2 proteins which comprise culturing a host cell transformed with an expression vector and causing expression of the nucleic acid encoding the cathepsin O2 protein to produce a recombinant cathepsin O2 protein.
A further aspect of the present invention provides poly- and monoclonal antibodies to cathepsin O2 proteins.